Structure and Function of Yeast Small Nuclear RNPs

说明书(由申请人提供)：SnRNA结构元件与其在剪接体组装和剪接中的功能的关系仅被一般理解。剪接中一个长期存在的谜团是许多RNA解旋酶家族成员是如何在剪接体的组装、催化和拆卸过程中实现SnRNA重排的。第二个主要问题涉及剪接调控因子如何影响剪接体组装的基本过程。许多调节剪接的蛋白质是已知的，但它们到底是如何起作用的仍然相当不清楚。第三个也是正在出现的一组问题涉及剪接和其他小RNA介导的过程在基因组功能和进化中的作用。我们将有一个明确的目标来解决这些主要问题。(1)分析了在体内外剪接体组装和剪接过程中，DEAD-box RNA解旋酶家族成员PrpSp与U2 SnRNP组分，特别是Cus2p和U2 SnRNA之间的功能相互作用。Cus2p和PrpSp影响U2 SnRNA结构，并相互影响，控制U2 SnRNA在ATP依赖的步长前体组装中将U2 SnRNA运送到Pre-mRNA的分支点区域。(2)我们将探索Mer1p激活剪接的机制及其在酵母减数分裂基因表达程序中的重要性。由于Merlp与U1 snRNP结合，因此我们将重点研究在Mer1p和不存在Mer1p的情况下U1 snRNP的组成和活性。目的的第二部分是了解Mer1p和四个已知基因的作用，这些基因的剪接依赖于Mer1p在利用剪接敏感微阵列的减数分裂基因表达程序中。(3)我们将研究剪接与基因表达途径中其他步骤的整合，并将开始确定含有保守序列的内含子的丢失如何影响细胞功能。上个资助期完成的一项大型微阵列研究提出了几个关于剪接和小RNA如何与基因表达中的其他步骤相联系的具体假设。这些目标所代表的研究领域涵盖了剪接领域的主要问题：SNRNP如何工作？拼接监管是如何实现的？剪接过程是如何整合到基因组功能和进化中的？酵母中可利用的遗传学、生物化学和基因组学的综合优势以及剪接机制的保守特性表明，通过这些努力发现的基础知识将直接转化为对人类基因表达的理解。

DESCRIPTION (provided by applicant): The relationships of the snRNA structural elements to their functions in spliceosome assembly and splicing are only generally understood. A persistent mystery in splicing is how the many RNA helicase family members achieve snRNA rearrangements during assembly of the spliceosome, catalysis, and disassembly. A second major question concerns how splicing regulators influence the basic process of spliceosome assembly. Many proteins that regulate splicing are known, but exactly how they do it remains quite opaque. A third and emerging set of questions concerns the role of splicing and other small RNA-mediated processes in genome function and evolution. We will address each of these major questions with a specific aim. (1) We will dissect functional interactions between the DEAD-box RNA helicase family member PrpSp and components of the U2 snRNP in particular Cus2p and U2 snRNA during spliceosome assembly and splicing in vitro and in vivo. Cus2p and PrpSp influence U2 snRNA structure and each other to control the delivery of U2 snRNA to the branchpoint region of the pre-mRNA in an ATP-dependent step-prespliceosome assembly. (2) We will explore the mechanism of Mer1p-activated splicing and its importance for the meiotic gene expression program in yeast. Since Merlp binds U1 snRNPs, we will focus on the composition and activities of the U1 snRNP with and without Mer1p. A second part of this aim is to understand the roles of Mer1p and the four known genes whose splicing depends on Mer1 p in the meiotic gene expression program using splicing sensitive microarrays. (3) We will study the integration of splicing with other steps in the gene expression pathway and will begin to determine how the loss of introns containing conserved sequence affects cellular function. A large microarray study completed in the last funding period presents several specific hypotheses for how splicing and small RNAs are connected to other steps in gene expression. The areas of inquiry represented by these aims encompass the major issues in the splicing field: How do snRNPs work? How is splicing regulation achieved? And how is the process of splicing integrated into genome function and evolution? The combined strengths of genetics, biochemistry, and genomics available in yeast and the conserved properties of the splicing machinery indicate that fundamental knowledge uncovered by these efforts will translate directly to the understanding of human gene expression.